Natural products from medicinal plants in Asia and the Pacific for RNA viruses: Hercules’ fifth labour

Abstract Context The emergence of zoonotic viruses in the last decades culminating with COVID-19 and challenges posed by the resistance of RNA viruses to antiviral drugs requires the development of new antiviral drugs. Objective This review identifies natural products isolated from Asian and Pacific medicinal plants with in vitro and in vivo antiviral activity towards RNA viruses and analyses their distribution, molecular weights, solubility and modes of action. Materials and methods All data in this review was compiled from Google Scholar, PubMed, Science Direct, Web of Science, ChemSpider, PubChem and library search from 1961 to 2022. Results Out of about 350 molecules identified, 43 phenolics, 31 alkaloids, and 28 terpenes were very strongly active against at least one type of RNA virus. These natural products are mainly planar and amphiphilic, with a molecular mass between 200 and 400 g/mol and target viral genome replication. Hydroxytyrosol, silvestrol, lycorine, tylophorine and 12-O-tetradecanoylphorbol 13-acetate with IC50 below 0.01 µg/mL and selectivity index (S.I.) above 100 have the potential to be used for the development of anti-RNA virus leads. Discussion and conclusions The medicinal plants of Asia and the Pacific are a rich source of natural products with the potential to be developed as lead for the treatment of RNA viral infections.


Introduction
Waves of zoonotic RNA virus pandemics have been infecting humans for about the last 100 years starting with the Spanish flu of 1918, followed more recently by the Middle East respiratory syndrome coronavirus (MERS-CoV; with a mortality rate of 35.4%) and since December 2019, COVID-19 (coronavirus disease 2019) caused by the severe acute respiratory virus-associated coronavirus 2 (SARS-CoV-2) (Ye et al. 2020). Although vaccines afford good protection against these viruses, populations are left without effective chemotherapeutic defence until vaccines are developed; as a result, there is a dramatic need to find antiviral lead compounds to develop into innovative antiviral drugs. Such a wonder molecule could come from nature, and in particular, the flowering plants of Asia and the Pacific, which are the oldest, largest and richest on Earth.
Flowering plants also called Angiosperms are organised into 11 major taxa or Clades distributed into three groups: (i) Basal Angiosperms including Protomagnoliids, Magnoliids, Monocots and Eudicots; (ii) Core Angiosperms covering the Core Eudicots, Rosids, Fabids and Malvids, and the Upper Angiosperms that include the Asterids, Lamiids and Campanulids (Haston et al. 2009). Within each Clades, plants yield specific types of secondary metabolites to control phytopathogenic microbes including viruses. These antimicrobial natural products fall into two main groups: phytoanticipins and phytoalexins. Phytoanticipins are antimicrobial compounds that are present before the challenge by phytopathogenic microbes, such as tannins or inactive immediate precursors stored in healthy tissues as flavonoid glycosides which get converted into antimicrobial metabolites known as phytoalexins.
The discovery of antivirals from plant phytoalexins requires an understanding of viral replication which includes six stages (Samji 2009). These are: (i) attachment of virion to host cell, (ii) fusion and entry of virion with the cytoplasmic membrane of the host cell, (iii) uncoating of the virus and release of the viral contents in the host cell, (iv) building of new viral genome and macromolecules using the host cell, (v) assembly of viral genome and macromolecules into viral progeny, and (vi) release of viral progeny that can often induce host cell death. For RNA viruses to produce viral progeny, the viral genome must be translated, i.e., converted into proteins that will further associate with viral particles. Single-stranded (þ)-RNA genome of single-stranded (þ)-RNA viruses are translated directly by host cell ribosomes into a large protein (or polyprotein) that is cleaved by viral proteases into structural proteins and enzymes that catalyses the synthesis of a complementary strand of virus-specific RNA. If the single-stranded (þ)-RNA viruses are equipped with reverse transcriptase, such as the Human Immunodeficiency virus (HIV), a complementary single-stranded DNA is produced which is further converted into double-stranded DNA that integrates into the host DNA permanently. Human pathogenic (þ) RNA viruses are found in the family Coronavidae, such as SARS-CoV-2, Flaviviridae, such as the Dengue virus (DV) and Picornaviriae, such as the Human Rhinovirus. Single-stranded (À)-RNA genome is replicated by the host cell and the viral RNA-dependent RNA polymerase to produce single-stranded (þ)-RNA that serves as a messenger RNA as well as intermediate for further synthesis of virus-specific genome (À)-RNA. Likewise, for double-stranded RNA viruses, RNA-dependent RNA polymerase uses the negative strand of the double-stranded RNA to generate a single-stranded (þ)-RNA. Human pathogenic (À)-RNA viruses are found in the family Paramyxoviridae and Orthomyxoviridae, such as the influenza virus.
The search for natural products that inhibit the replication of RNA viruses also require the measurement of the antiviral strength in vitro that is quantitively based on the IC 50 and selectivity index (S.I.). Ideal candidates have low IC 50 and large S.I. Consider that natural products from plants with antiviral activity have often some levels of cytotoxicity. Hence, a good antiviral property can be observed in vitro and at the same time toxicity towards cultured host cells. The generally accepted threshold of S.I. in the area of antiviral development is 10, i.e., if a compound demonstrates S.I. of 10 or higher it is considered prospective (Smee et al. 2017).
This review addresses the following points in regard to the natural products with anti-RNA virus activity identified from the flowering plants of Asia and the Pacific: distribution, strength, spectrum of activity, influence of molecular mass and solubility, mechanisms of action, structure-activity, selectivity index, in vivo activity and clinical potentials. For about the last 80 years, an enormous body of experimental evidence has been generated with the hope to find anti-RNA virus natural products of clinical value in plants globally. This review provides a taxonomical, phytochemical, biomolecular and physicochemical rationale to facilitate the discovery of leads for the treatment of RNA virus infections.

Enterovirus 71 (EV-71)
EV-71 accounts for the hand, foot, and mouth disease that has the potential to develop into life-threatening neurological disorders in young children. The virion surface VP1 protein binds to the scavenger receptor B2 and human P-selectin glycoprotein ligand-1 of host cells and the viral (þ)-RNA is used for the synthesis of a polyprotein (Kuo and Shih 2013). A number of natural products are very strong suppressors of this virus (Table 1).

Coxsackievirus (CV)
CV accounts for myocarditis. The virion binds to the decay accelerating factor (DAF/CD55) and viral (þ)-RNA is translated into a polyprotein further cleaved into structural and non-structural proteins by viral proteases 2 A pro and 3 C pro . No treatment is available. A number of natural products are very strong suppressors of this virus (Table 1).

Encephalomyocarditis virus (EMCV)
EMCV is zoonotic from rodents and accounts for fatal neurological and cardiac insults in apes, pigs, and human (Oberste et al. 2009). No treatment exists for this virus. The virion follows a classic cell cycle including cleavage of polyprotein by viral 3CProtease (3 C Pro ). The iridoid glycoside arbortristoside A in Nyctanthes arbor-tristis L. (Oleaceae) strongly suppressed EMCV.

Porcine reproductive and respiratory syndrome virus (PRRSV)
PRRSV accounts for respiratory infections and sterility of unvaccinated pigs. The virion binds to the sialo-adhesin receptor of macrophages and release a single-stranded (þ)-RNA translated further into a polyprotein that is cleaved into structural proteins and enzymes, the latter catalysing the synthesis of a doublestranded RNA (Wang, Dong, et al. 2021;Wang, Zeng, et al. 2021). Phenolics in C. sinensis repressed PRRSV at the concentration of 10 mg/mL (Wang, Dong, et al. 2021;Wang, Zeng, et al. 2021) and the kaurane diterpene methyl pothoscandensate is a weak inhibitor (Liu et al. 2012).

Transmissible gastroenteritis virus (TGEV)
Another a-coronavirus infecting pigs is TGEV which is very strongly inhibited by the phenanthroindolizidine alkaloid tylophorine (20) in plants from the genus Ficus L. (Moraceae) with an IC 50 value as low as 58 nM and a S.I. above 1715 (Yang, Lee, et al. 2010;Yang, Li, et al. 2010).
Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) SARS-CoV is responsible fever, cough and breathing difficulties and originated from the consumption of masked palm civets and racoon dogs in China (Ye et al. 2020).

Sinigrin
Severe acute respiratory syndrome-associated coronavirus À 2 (SARS-CoV-2) SARS-CoV-2 may have originated from the consumption of pangolin or bats in China and is responsible for life-threatening pneumonia Ye et al. 2020).

Terpenes
Artemisia vulgaris L. (Asteraceae) yields the sesquiterpene artemisinin that given to COVID-19 patients at 125 mg with piperaquine 750 mg on the first day followed artemisinin 62.5 mg and piperaquine 375 mg/d for 6 d evoked clearance of the virus and levels of pulmonary protection (Li et al. 2021). Andrographis paniculata (Burm. f.) Wall. ex Nees (Acanthaceae) produces the labdane diterpene andrographolide which when given orally to COVID-19 patients at the dose of 20 mg 3 times per day for 5 d prevented the development of pneumonia (Wanaratna et al. 2021). The cardiac glycoside oleandrin (74) from Nerium oleander L. (Apocynaceae) yielded an EC 50 value of 11.9 ng/mL (Plante et al. 2021). Oleandrin (74) given to hamsters (sub-lingually at the dose of 25 mL of a 130 mg/mL solution once daily for 5 d) evoked some levels of protection (Plante et al. 2021).

Avian infectious bronchitis virus (IBV)
IBV evokes in unvaccinated chicken and turkey bronchitis, cough, fever, conjunctivitis and fatal respiratory distress. The spike proteins bind to sialic acid and the virus is internalised to release a single-stranded (þ)-RNA that is transcribed into structural proteins and enzymes. Perusal of literature indicates that natural products in medicinal plants in Asia and the Pacific are meek repressors of IBV in general such as forsythoside A  or b-pinene .

Dengue virus (DV)
DV is responsible for fever and bleeding. The virion binds via the envelop protein E to heparan sulphate on the surface of host cells . Very strong inhibitors of DV in vitro are Amaryllidaceae alkaloids (Table 1).

Lignans
The neolignan honokiol from Magnolia officinalis Rehder and E.H. Wilson (Magnoliaceae) is a strong repressor of DV (Fang et al. 2015).

Japanese encephalitis virus (JEV)
JEV accounts for fatal central nervous system damage. The virion E protein binds to the host cell LDL-receptor and the rest of the cycle is similar to as DV (Yun and Lee 2014). Very strong repressors of JEV are Amaryllidaceae alkaloids (Table 1).

Zika virus (ZIKV)
ZIKV is a zoonotic virus transmitted by mosquitoes and responsible for outbreaks of fever, rashes, conjunctivitis, foetal anomalies, myalgia and in some patients sever neurological disorder. The virion surface protein E binds to lectins and after internalisation, (þ)-RNA is translated into a polyprotein cleaved by viral protease complex NS2B-NS3 (Sager et al. 2018).

Phenolics
The strongest inhibitors of ZIKV within the phenolics group are lignans, such as silvestrol (8)

Alkaloids
Hippeastrine from Lycoris radiata (L 0 H er.) Herb. (Amaryllidaceae) inhibited ZIKV with an IC 50 value of 1.9 lM and given subcutaneously at a dose of 100 mg/kg/d to mice evoked some levels of protection . Palmatine and glycyrrhizin are weakly active (Crance et al. 2003).

Hepatitis C virus (HCV)
HCV is transmitted by blood via transfusion or sex and account for hepatocellular carcinoma in humans. The virion surface glycoprotein E binds to heparan sulphate of hepatocytes and (þ)-RNA is translated into a polyprotein cleaved into structural proteins and enzymes, such as HCV NS5B polymerase (Bhatia et al. 2014).

Stilbenes
The oligostilbenoid E-viniferin repressed HCV with the EC 50 value of 0.1 lM ).

Mayaro virus (MAYV)
MAYV is transmitted from primates to humans via mosquitoes and accounts for rashes, fever, myalgia, retro-orbital pain, headache, diarrhoea and long-lasting arthralgia (Esposito et al. 2016). The virion binds to heparan sulphate to release a (þ)-RNA that is directly translated by host ribosome into a polyprotein later cleaved into functional proteins (Mota et al. 2015). Epicatechin (35) strongly repressed MAYV (strain Bear 20290) with the IC 50 value of 0.2 mM (Ferraz et al. 2019).

Human immunodeficiency virus (HIV)
HIV is a zoonotic virus transmitted with blood from apes to humans and responsible for AIDS (De Clercq 2007). This virus target CD4 cells resulting in a decrease of immunity, which in most untreated patient, leads to fatality. The viral glycoprotein 120 binds to the CCR5 and CXCR4 receptor and viral RNA is translated to DNA by viral reverse transcriptase (Zack 1995). A broad array of natural products is able to repress HIV in vitro (Table 1).
(2) RNA viruses Newcastle disease virus (NDV) NDV incurs fever, diarrhoea, anorexia and fatality in unvaccinated poultry. The surface haemagglutinin-neuraminidase glycoprotein of the virion binds to host cell sialic acid and the single-stranded (-)-RNA is translated by the viral RNAdependent RNA polymerase into a (þ)-RNA used for the synthesis of proteins by host ribosomes (Zaitsev et al. 2004).

Measles virus (MEV)
MEV is fatal for unvaccinated children especially in developing countries. The virion binds via its surface haemagglutinin-neuraminidase glycoprotein to CD46 or PVRL4 host cell receptors and after capsid internalisation the single-stranded (-)-RNA is translated by viral RNA-dependent RNA polymerase into a (þ)-RNA used for the synthesis of protein by host ribosomes (Bhattacharjee and Yadava 2018). The naphthoquinone droserone in Drosera peltata Thunb. (Droseraceae) is moderately active against this virus (Lieberherr et al. 2017) as well as angeloyl heliotrine (Singh et al. 2002) and the dammarane triterpene saponin chikusetsusaponin IV a from Alternanthera philoxeroides (Mart.) Griseb. (Amaranthaceae) (Rattanathongkom et al. 2009).

Respiratory syncytial virus (RSV)
RSV is responsible for cold in adults but cause life-threatening pneumonia in young infants globally. The virion binds to heparan sulphate on the surface of host cell, enters the cell and after capsid disintegration the single-stranded (-)-RNA is released in the cytoplasm where viral RNA-dependent RNA polymerase transcribes it into (þ)-RNA (Griffiths et al. 2017).

Lassa virus (LASV)
LASV is transmitted from rats and accounts for fatal haemorrhages (Reuben et al. 2020). The amide alkaloid capsaicin from Capsicum annum L. (Solanaceae) moderately repressed this virus .

Influenza virus (IV). Influenza virus (IV)
is zoonotic from aquatic birds and evokes fever, cough and shortness of breath that if not treated in unvaccinated elderly patients may eventually lead to fatal pneumonia. The viral surface haemagglutinin and neuraminidase glycoproteins binds to the host cell sialic acid and after internalisation and disintegration of viral capsid, single-stranded (-)-RNA is translated by viral RNA-dependent RNA polymerase into a (þ)-RNA used for the synthesis of protein by host ribosomes (Samji 2009). Perusal of literature indicates that very strong IV inhibitors are mainly phenolics (Table 1).

Alkaloids
Berberine (99) yielded the IC 50 value of 0.01 mM and given intraperitoneally to mice, at a dose of 5 mg/kg/d for 7 d decreased influenza related mortality rate from 90% to 55% in rodent (Shao et al. 2020). Other isoquinolines of interest are lycorine (3) (Tietze and Zhou 1999). The quinazoline alkaloid camptothecin is active against IV (Kelly et al. 1974) as well as melicopteline C , dendrobine (IC 50 2.1 mg/mL)  and homonojirimycin .

The distribution of anti-RNA virus natural products among the various clades of angiosperms in Asia and the Pacific
Regarding the distribution of anti-RNA natural products (Tables  2 and 3), the following observations can be made: (i) All Clades except the Protomagnoliids yield inhibitors of both (þ) and (À)-RNA viruses; (ii) The strongest inhibitors of RNA viruses are mainly found in Basal Angiosperms; (iii) Some Clades yield specific antiviral natural products, such as dibenzocyclooctadiene lignans in the Protomagnoliids, oligostilbenes in the Rosids or quinolizidines and quinolizidines and isoflavones in the Fabids, naphthylisoquinoline alkaloids in the Malvids, monoterpene indole alkaloids and quinazoline alkaloids in the Lamiids; (iv) Anti-RNA virus phenolics, flavonoids and triterpenes are in general widespread; (v) Basal Angiosperms use mainly sesquiterpenes, lignans and isoquinolines as phytoalexins; (vi) Core Angiosperm use mainly phenolics as phytoalexins; (vii) Upper Angiosperm use mainly hydroxycinnamic acid derivatives, iridoid glycosides, indole alkaloids, diterpenes, triterpene saponins and acetylenic fatty acids as phytoalexins; (viii) Very strong anti-RNA natural products occur mostly in medicinal plants used for microbial infection (Table 4).

The strongest anti-RNA virus natural products from the flowering plants of Asian and the Pacific
Compared with antibacterial or antifungal principles from plants, no accepted classification criteria for in vitro strength of antiviral activity seem to be available. We therefore suggest for a compound: (i) IC 50 value below or equal to 1 mg/mL ¼ very strongly active, (ii) for an IC 50 value above 1 and equal to or below 20 mg/mL ¼ strongly active, (iii) for an IC 50 above 20 and below or equal to 100 mg/mL ¼ moderately active, (iv) for an IC 50 above 100 and below or equal to 500 mg/mL ¼ weakly active, (v) for an IC 50 ranging from above 500 to below or equal to 2500 mg/mL ¼ very weakly active and, inactive, an IC 50 values above 2500 mg/mL. Accordingly, out of about the 350 molecules characterised, 102 (43 phenolics, 31 alkaloids and 28 terpenes) are very strongly active against at least one type of RNA virus (Table 1, Figure 1). The lowest IC 50 values were achieved by oleandrin (74) (Plante et al. 2021), silvestrol (8) (M€ uller et al. 2018;Henss et al. 2018), pseudolycorine (23) and lycoricidine (24), pancratistatin (25), narciclasine (26), stelleracin A (67), B (68), and C (69), quercetin (91) (Ibrahim et al. 2013), rutin (92), (Ibrahim et al. 2013) and hypericin (96) (Yasuda et al. 2010).

Spectrum of activity
As for the spectrum of activity, the following observation are made: (i) Tannins and saponins are not very strongly active   against non-enveloped viruses suggesting that they tend to act by inhibiting viral anchoring and fusion (non-enveloped viruses do not undergo fusion); (ii) A few natural products very strongly repress non-enveloped (þ)-RNA viruses; (iii) None of the alkaloids identified showed strong activity against enveloped, monopartite, linear, single-stranded (-)-RNA viruses; and (iv) silvestrol (8), lycorine (3) and lycoricidine (24) inhibit a broad spectrum of RNA viruses. The influence of molecular mass The molecular mass of natural products tends to influence their ability to fit in the catalytic pockets of enzymes and to cross biological membranes. Here, we define low molecular mass molecules with a molecular mass below 200 g/mol, medium molecular mass molecules with a molecular mass from 200 to 400 g/mol, and high molecular mass molecules with a molecular mass above 400 g/mol (Table 1). Following this classification, we note that principles with very strong activity have mainly a medium to high molecular mass.
The influence of solubility: the importance of amphiphilicity The water solubility of natural products dictates their ability to cross biological membranes. For instance, lipophilic molecules and to a lesser extend amphiphilic molecules can penetrate cells via passive diffusion whereas large hydrophilic molecules require active transports or pinocytosis (Rustad 1961). Log P is equal to the ratio of concentrations of a compound between octanol and water. Hydrophilic compounds (hydrophilic) have low or negative values (about À3) (compounds are mainly found in the water phase). Mid-hydrophilic compounds have Log P is near to 0 (the compound is equally partitioned between the octanol and water layers). Non-hydrophilic (hydrophobic, liposoluble) compounds have a high Log P (up to about 7) (note that lipophilic natural products may tend to remain in and to destabilise the outer membrane of enveloped viruses and/or host cells). However, Log P is only relevant for non-ionizable principles and for ionised substance (such as berberine (98) has a Log D is preferable at pH 7.4. Here we define at pH 7.4 hydrophilic compounds for a negative LogD to a value 1, amphiphilic (mid-polar) compounds for a LogD above 1 up to about 4.5, and lipophilic for a LogD above about 5. Note that LogD values given here are predicted values. Accordingly, molecules with very strong activity are mainly hydrophilic or amphiphilic.

Mechanism of action: non-specific and/or specific targets
Natural products from the medicinal plants of Asia and the Pacific have, depending on their chemical structure, non-specific and or specific mechanism of annihilation of RNA viruses.

Inhibitors of virus fusion and entry
Natural products inhibiting virus attachment to host cells repress virus internalisation, such as tetra-O-galloyl-b-D-glucose (Yi et al. 2004). Agents blocking viral fusion are phenolics such as (þ)-catechin gallate   Trachelogenin (31) inhibited HCV entry in Huh7 cells by blocking glycoprotein E2 binding to CD81 (Qian et al. 2016). Capsaicin blocked LASV entry in host cells (Tang, Song, et al. 2020;. Regarding saponins, they tend to block the entry of HIV in host cells as seen with arganine (Gosse et al. 2002) and cimicifugin with RSV . The precise mode of antiviral action of saponin is unknown but they may alter the outer envelope of enveloped viruses and  compromise the anchoring of virus to host cells. Further, saponins bind to membranes cholesterol. The viral envelope contains cholesterol which promotes the fusion of viruses with host cells, and cholesterol in the host cell membrane promotes the fusion of the virion to the host cell (Ahn et al. 2002). Since saponins bind to membrane cholesterol it is possible to propose an anti-RNA virus mode of action of triterpene saponins and saponins at large which could involve, at least in part, binding to viral and host membrane cholesterol. In this light, it can be suggested that non-enveloped RNA viruses resist phenolics, saponins, and tannins because they are deprived of outer membranes. As for tannins, geraniin (43) suppressed HIV-1 entry in MT4 cells (Notka et al. 2003).
Cardiac glycosides such as ouabain and digitoxin are broadspectrum antiviral molecules acting on the replication of the CHIKV, MERS and SARS-CoV via inhibition of Na þ /K þ -ATPase (Amarelle and Lecuona 2018;Yang et al. 2018). Inhibition of Na þ /K þ ATPase by oleandrin (74) attenuates ZIKV infection in mice. Conessine inhibited ZIKV (strain PAN2016) (IC 50 9.7 mM) (Lima et al. 2021) via ion channel inhibition. Kuranone inhibits virus-induced autophagic flux (Min et al. 2020). Ion channels are involved in the viral internalisation of several viruses including EBOV, and oleandrin (74), ouabain, digitoxin and other cardiac glycosides which block Na þ / K þ ATPase suppress a broad spectrum of viruses (Amarelle and Lecuona 2018). Disturbance of host cell cytoplasmic membrane potential by blocking ion channels (Charlton et al. 2020) may account for the activities of alkaloids such as conessine (Lima et al. 2021;Shen et al. 2019). Conessine binds to G-protein coupled receptors and ligand-activated ion channels.
MERS-CoV translocates in the endolysosomal system of host cells via a mechanism involving Ca 2þ -permeable channels. SARS-CoV encodes for 3a protein which forms ion channels that become incorporated into the membrane of the host cells. Flavonol glycosides inhibit 3a protein in vitro, such as tiliroside, kaempferol 3-Oarabinoside and afzelin. Berbamine is a calcium channel blocker that inhibits late-stage of Flavivirus replication Chen, Yang, Zhai, et al. 2020). Tetrandrine and fangchinoline (21) impair MERS-CoV translocation in the host cell and are known channel inhibitors (Charlton et al. 2020). Hence, natural products known for being calcium channel inhibitors have the potential to be anti-coronavirus agents. In coronaviruses, calcium-dependent nucleocapsid and non-structural protein 3 (nsp3) interactions are necessary for replication. Further, Fujioka et al.
(2018) noted that IVA haemagglutinin binds a sialylated voltagedependent calcium channel Cav1.2 to trigger increased cytoplasmic calcium and subsequent Influenza virus entry and replication. It can be inferred that disturbance of membrane potential can induce changes in cytoplasmic calcium homeostasis whence blockage of viral entry. Thus, calcium modulators, which are manifold among natural products from Asian medicinal plants need to be examined. One such modulator is palmatine which blocks ZIKV entry in Vero cells.
Inhibitors of uncoating of the virus and release of the viral contents in the host cells These are apparently few but ion channels are involved in pH modification of endosomes resulting in capsid disintegration in host cells suggesting that ion channels inhibitors have the potential to be antiviral.

Translocation of naked virion to host cell nucleus
Podophyllotoxin, a well-known mitotic spindle poison of therapeutic value inhibits tubulin incorporation into microtubules. Consider that microtubules act as 'anchor' for viral capsid proteins to allow processes such as transport of naked virion to the nucleus, mRNA transcription, DNA replication and DNA packaging prior to virion release (Waye and Sing 2010) and it could be inferred that tubulin poisons have the potential to be developed into antiviral agents. Tubulin poisons among flowering plants in Asia and the Pacific include apocynaceous monoterpene alkaloids.

Topoisomerase inhibitors
Planar heterocyclic indole alkaloids intercalate DNA and block topoisomerase. Topoisomerase is needed for (þ)-RNA virus replication. The most active antiviral alkaloids are planar and as such may interact with viral DNA or DNA-processing enzymes such as topoisomerase . Manassantin B inhibits topoisomerase ) and this mechanism could explain why it inhibits the replication of CV and HIV. Camptothecin stabilises topoisomerase I-DNA complex and inhibits the religation of DNA and therefore blocks virus replication. Protoberberines are DNA-stabilizing agent and this may explain their antiviral effects. In this light, carbazole alkaloids could be screened for their antiviral activities.

Polymerase inhibitors
RNA-dependent RNA polymerase catalyses the synthesis of a double-stranded RNA replicated into multiple (þ)-RNA. This enzyme is a therapeutic target of drugs such as Remdesivir and is inhibited by phenolics, such as robinetin (Ahmed-Belkacem et al. 2014), myricetin (Ono et al. 1990), and usnic acid (Peyrat et al. 2016). Sennosides A and B strongly inhibited the HIV DNA polymerase (Esposito et al. 2016) as well as Amaryllidaceae alkaloids. Examples of terpenes inhibiting HIV DNA polymerase are pomolic acid, koetjapic acid (Sun et al. 1999), and gossypol. Platycodin D (33), platycodin D2 and platycodin D3 inhibited HCV NS5B polymerase . Natural products inhibiting RNA replication have mainly medium molecular masses and are principally amphiphilic.

Ribonuclease inhibitors
These are anthraquinones such as rhein, sennosides A and B (Esposito et al. 2016) and naphthoquinones such as juglone .

Protein kinase inhibitors
The transcription of viral genetic material by enveloped viruses including a-viruses is regulated by phosphorylation mediated by the virus-associated protein kinase. Berberine (98) inhibited of mitogen-activated protein kinase signalling in host cells infected by CHIKV (Varghese et al. 2016). Inhibition of protein kinase C increases host cell death infected by SINV in vitro and we argue that the antiviral activity of 12-O-tetradecanoylphorbol 13-acetate (34) and other tiglianes towards viruses is due, at least in part, to protein kinase C activation. The protein Gag mediates the assembly and release of virus from an infected host cell and this mediation requires its phosphorylation by host cell protein kinase C and this may explain, at least in part, why natural products inhibiting protein kinase C such as tigliane diterpenes are often antiretrovial ).

Assembly of viral genome and macromolecules into progeny virions
Berberine (98) repressed IV protein trafficking/maturation.

Release of viral progeny
The release of newly formed HIV virus from host cell requires cleaving of Gag protein by a viral protease (Freed 2015) which is blocked by ellagic acid (Modi et al. 2013). In (-)-RNA viruses, neuraminidase catalyses the hydrolysis of the a-(2,3)or a-(2,6)glycosidic linkage between a terminal sialic acid residue and its adjacent carbohydrate moiety on the host receptor allowing the release from hots cells of the newly formed viral progeny, and Oseltamivir is an example of neuraminidase inhibitor used for the treatment of influenza (Moscona 2005).

Structure-activity: the importance of planarity
In general, the more planar is a secondary metabolite of medium molecular mass the more specific the antiviral activities due to, at least in part, their ability to intercalate into viral genetic material and subsequent inhibition of enzymes targeting DNA and/or RNA. Regarding phenolics, the following observations can be made: (i) the presence of a quinone moiety boosts the activity of phenolics such as aloe-emodin (2) (Lin et al. 2008), 2-(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-dione (Takegami et al. 1998), shikonin (Lin et al. 2008), plumbagin (32) as well as naphthalene framework such as diphyllin (30) (Cui et al. 2014) or hypericin (96) (Yasuda et al. 2010); (ii) Glycosylation of flavonols is detrimental for activity as seen in pectolinarin (Simões et al. 2011) and quercetrin (Chiow et al. 2016). Glycosylation of stilbenes translates in weaker potencies against IV such as piceid (Nguyen et al. 2011); (iii) Oligomerization of stilbenes is not detrimental to antiviral activity (Yang et al. 2005) neither is the condensation of flavonols in bisflavonols (de Freitas et al. 2020); (iv) Methoxylation of flavonols is detrimental to activity ); (v) Introduction of ketone group in position 3 of flavans increases anti-HIV activity as seen with kuwanon L. (EC 50 1.9 mM) (Esposito et al. 2015). Methoxylation of isoorientin is detrimental to activity against RSV . Planar indole alkaloids are often strongly retroviral (49) (Hsieh et al. 2004) via topoisomerase inhibition . Strong PL pro inhibitors are mainly planar (Park et al. 2012). Opening of the methylenedioxy moiety of Amaryllidaceae alkaloids is detrimental to activity towards DV (Zou et al. 2009).

Natural products active in vivo
Natural products with very strong activity in vitro are seldom active in vivo orally because of first-pass metabolism. In the case of flavonoids and hydroxycinnamic acids and especially saponins, oral bioavailability is in general modest, however, oral activity was observed with chlorogenic acid, forsythoside A (Law et al. 2017), cinnamic acid (Hayashi et al. 2007), tangeretin (Xu et al. 2015), isoorientin, homonojirimycin ) and polyphylla saponin I (Pu et al. 2015). Berberine (98) has low oral bioavailability and afforded levels of protection in mice infected with IVA (Shao et al. 2020). An example of sesquiterpene active in vivo orally is atractylon . Other instances are calycosin-7-O-b-D-glucopyranoside (Zhu et al. 2009), manassantin B (Song et al. 2019) and gymnemic acid A (Sinsheimer et al. 1968).

Concluding remarks
Compared with currently available antiviral drugs, natural products from Angiosperms in Asia and the Pacific appear to have numerous targets acting synergistically and at the various stages of RNA virus replication. The mechanisms of action of these principles in vitro are also dependent on their concentrations. Weinstein and Albersheim (1983) presented evidence that natural products from Angiosperms act non-specifically in order to avoid the development of resistance by phytopathogenic microbes (Amoros et al. 1992). This is one of the reasons why isolating an antiviral natural product from Angiosperms for clinical systemic use with a low therapeutic index and working at micromolar plasmatic concentration is an Augean task. Some natural products have reached clinical trials for HIV such as calanolide A (41) and others, like shikimic acid, are used as the starting material for the synthesis of antiviral drugs . It is, therefore, reasonable to anticipate the identification of a lead for the prevention and/or treatment of RNA virus infection from the medicinal Angiosperms of Asia and the Pacific. Hydroxytyrosol (36), silvestrol (8), lycorine (3), tylophorine (20) and 12-O-tetradecanoylphorbol 13-acetate (34) with IC 50 values below 0.01 mg/mL and S.I. above 100 (or their hemisynthetic derivatives) may have the potential to be developed as anti-RNA virus leads. In the rural settings of Southeast Asia and the Pacific, medicinal plants are used as herbal remedies for the treatment of COVID-19 and other viral diseases and some of them with proven efficacy, such as A. paniculata (Wanaratna et al. 2021) or B. rotunda. Another alternative would the development of extracts of fractions undergoing clinical trials and manufactured under strict pharmaceutical control. In this light, it is of the uttermost importance to preserve the primary rainforests of Asia and the Pacific and the medicinal traditional knowledge of indigenous tribes.

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Funding
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